The most efficient and well developed systems for in vivo gene therapy are based upon retroviruses because the use of retroviral vectors (RV) is currently the method of choice for the transfer of therapeutic genes in a variety of approved protocols both in the USA and in Europe (Kotani, H., et. al., Human Gene Therapy 5: 19-28(1994)). Retroviruses infect a wide variety of cells and are ideal tools for the stable delivery of genes to cells since the retrovirus is able to integrate the DNA form of its genome into the genome of the target cell. Thus, all daughter cells of an infected cell carry the retroviral vector DNA including the therapeutic gene. However, current efficient transfer of therapeutic genes usually requires that the infection of target cells with the RV carrying the genes occurs in vitro, and successfully infected cells are then returned to the affected individual (Rosenberg, S. A., et. al., Hum. Gene Ther. 3:75-90(1992); Anderson, W. F., Science 256:808-813(1992)). Such ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated. Additionally, the ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use.
Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated, cultured and then reintroduced. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the RV, or cells producing the recombinant virus, are directly administered to the patient in the form of an injection or simple implantation of RV producing cells.
This kind of in vivo approach, of course, introduces a variety of new problems. First of all, and above all, safety considerations have to be addressed. Virus will be produced, possibly from an implantation of virus producing cells, and there will be no opportunity to precheck the produced virus. It is important to be aware of the finite risk involved in the use of such systems.
To minimize this risk the so-called ProCon vector has been developed as a safe gene transfer vehicle for targeted gene therapy using the principle of promoter conversion typical for retroviruses (PCT/EP95/03445):
The retroviral genome consists of an RNA molecule with the structure R-U5-gag-pol-env-U3-R. During the process of reverse transcription, the U5 region is duplicated at the right hand end of the generated DNA molecule, whilst the U3 region is duplicated and placed at the left hand end of the generated DNA molecule. The resulting structure U3-R-U5 is called LTR (Long Terminal Repeat) and is thus identical and repeated at both ends of the DNA structure or provirus. The U3 region at the left hand end of the provirus harbours the promoter. This promoter drives the synthesis of an RNA transcript initiating at the boundary between the left hand U3 and R regions and terminating at the boundary between the right hand R and U5 region. This RNA is packaged into retroviral particles and transported into the target cell to be infected. In the target cell the RNA genome is again reverse transcribed as described above.
In the ProCon vector the right hand U3 region is altered, but the normal left hand U3 structure is maintained; the vector can be normally transcribed into RNA utilizing the normal retroviral promoter located within the left hand U3 region. However, the generated RNA will only contain the altered right hand U3 structure. In the infected target cell, after reverse transcription, this altered U3 structure will be placed at both ends of the retroviral structure.
The altered region carries a polylinker instead of the U3 region. Thus, any promoter, including those directing tissue specific expression can be easily inserted. This promoter is then utilized exclusively in the target cell for expression of linked genes carried by the retroviral vector. Accordingly, in the packaging cell line the expression of the retroviral vector is regulated by the normal unselective retroviral promoter contained in the U3 region. However, as soon as the vector enters the target cell promoter conversion occurs, and the therapeutic and/or marker genes are expressed from a tissue specific promoter of choice introduced into the polylinker. Not only can virtually any tissue specific promoter be included in the system, providing for the selective targeting of a wide variety of different cell types, but additionally, following the conversion event, the structure and properties of the retroviral vector no longer resemble that of a virus. This, of course, has extremely important consequences from a safety point of view, since other retroviral vectors readily undergo genetic recombination with the retroviral packaging construct and/or endogenous retroviruses to produce potentially pathogenic viruses. Promoter conversion vectors do not resemble retroviruses because they no longer carry U3 retroviral promoters after conversion thus reducing the possibility of genetic recombination.
Rather than a tissue specific promoter a regulatable promoter can be inserted in the polylinker, allowing the conditional expression of genes carried by the retroviral vector.
However, all current systems of controlling gene expression are not absolute, and all require the additional inconvenience of requiring the presence of an inducer or repressor substance or stimulus.
The invention comprises the following, alone or in combination:
A retroviral vector comprising one or more promoters inserted in antisense orientation within the 5xe2x80x2 LTR region and one or more coding sequences inserted in antisense orientation within the 3xe2x80x2 LTR region, both, the promoter as well as the coding sequence, inserted in such a way as to ensure that the promoter and the coding sequence become duplicated during the process of reverse transcription in a target cell and appear in the 3xe2x80x2 as well as in the 5xe2x80x2 LTR region of the resulting provirus in a fashion where the promoter is located upstream of the coding sequence allowing it to drive gene expression.
the retroviral vector as above, wherein said promoter is inserted within the U5 region of the 5xe2x80x2 LTR;
the retroviral vector as above, wherein said coding sequence is inserted within the U3 region of the 3xe2x80x2 LTR;
the retroviral vector as above, wherein said coding sequence comprises heterologous DNA;
the retroviral vector as above, wherein said coding sequence is selected from one or more elements of the group consisting of marker genes, therapeutic genes, antiviral genes, antitumour genes, cytokine genes and/or toxin genes;
the retroviral vector as above, wherein said promoter is a strong, constitutive promoter;
the retroviral vector as above, wherein said retroviral vector is replication-defective;
the retroviral vector as above, wherein said retroviral vector is based on a vector of the pLXSN family;
the retroviral vector as above, wherein said retroviral vector is based on a promoter conversion vector;
a recombinant retroviral vector system comprising a retroviral vector as above as a first component, and a packaging cell line harbouring at least one retroviral and/or recombinant retroviral construct coding for proteins required for said retroviral vector to be packaged;
a retroviral particle produced by transfecting a packaging cell line of a retroviral vector system as above with the retroviral vector as above;
a retroviral provirus produced by infection of target cells with a recombinant retroviral particle as above;
mRNA of a retroviral provirus as above;
RNA of a retroviral vector as above;
a host cell infected with a retroviral particle as above;
a pharmaceutical composition containing a therapeutically effective amount of a recombinant retroviral particle as above and/or a recombinant retroviral vector system as above;
a method for introducing homologous and/or heterologous nucleotide sequences into target cells comprising infecting the target cells with recombinant retroviral particles as above;
use of a recombinant retroviral vector as above and/or of a recombinant retroviral vector system as above and/or of a retroviral particle as above for producing a pharmaceutical composition for gene therapy.